Certain mycobacteria represent major pathogens of man and animals. For example, tuberculosis is generally caused in humans by Mycobacterium tuberculosis, and in cattle by Mycobacterium bovis, which may also be transmitted to humans and other animals. Mycobacteria leprae is the causative agent of leprosy. M. tuberculosis and mycobacteria of the avium-intracellulare-scrofulaceum group (MAIS group) represent major opportunistic pathogens of patients with acquired immune deficiency syndrome. M. pseudotuberculosis is a major pathogen of cattle.
Globally, tuberculosis is the leading cause of death in adults due to an infectious organism (Dolin, et al., Bull. World Health Organ. 72: 213-220 (1994)). It is estimated that 90 million new tuberculosis cases resulting in 30 million deaths can be expected during the last decade of this century (Raviglione, et al., JAMA. 273: 220-226 (1995)). The resurgence of tuberculosis in developing nations (Snider, et al., In Tuberculosis: pathogenesis, protection and control., B. R. Bloom, (ed.), ASM Press, Washington, D.C. p. 3-11 (1994)), the appearance of multi-drug resistant strains of Mycobacterium tuberculosis, and the problem of tuberculosis in the immunocompromised (Haas, D. W. and R. M. Des Prez, Amer. J. Med. 96: 439-450 (1994)) call for further study of the mycobacteria. More knowledge about the basic biology of the mycobacteria is needed in order to develop a deeper understanding of the pathogenesis of mycobacterial diseases. Furthermore, identification of biological processes specifically essential for the growth and development of mycobacteria will allow the rational design of drugs to inhibit those processes. The complex cell envelope of the mycobacteria is an outstanding feature of these organisms (Brennan, P. J. and H. Nikaido. Annu. Rev. Biochem. 64: 29-63 (1995)). The envelope is composed of a variety of complex lipids including the long chain mycolic acids, and unique polysaccharides such as arabinogalactan and arabinomannan (Besra, G. S. and D. Chatterjee, Tuberculosis: pathogenesis, protection, and control., B. R. Bloom, (ed.), ASM Press, Washington, D.C. p. 285-306 (1994)). These components contribute to the hydrophobic nature of the mycobacterial cell surface (McNeil, M. R. and P. J. Brennan. Res. Microbiology. 142: 451-463 (1994)), the low permeability of the mycobacterial cell envelope (Nikaido, H. and V. Jarlier. Res. Microbiology. 142: 437-442 (1994), and play a role in the immunological responses of the host to mycobacterial infections (Chan, J. and S. H. E. Kaufmann. Tuberculosis: pathogenesis, protection, and control., B. R. Bloom, (ed.), ASM Press, Washington, D.C. p. 389-415 (1994)).
A specific and important area of interest is the biosynthesis of the peptidoglycan, the innermost layer of the mycobacterial cell wall (Brennan, P. J. and P. Draper. Tuberculosis: pathogenesis, protection, and control., B. R. Bloom, (ed.), ASM Press, Washington, D.C. p. 271-284 (1994)). Peptidoglycan is present in virtually all bacteria providing shape and structural integrity. The peptidoglycan of mycobacteria differs in a few respects from that of other bacteria. In most bacteria the glycan backbone of the peptidoglycan is comprised of N-acetylmuramic acid and N-acetylglucosamine (Ghuysen, J. M., Bact. Rev. 32: 425-464 (1968)). In the mycobacteria the former is replaced by N-glycolylmuramic acid (Azuma, et al., Biochim. Biophys. Acta. 208: 444-451 (1970). The peptide portion of mycobacterial peptidoglycan is of the common Alg chemotype, consisting of L-Ala-D-Gln-meso-diaminopimelate (meso-DAP)-D-Ala (Schleifer, K. H. and O. Kandler, Bac. Rev. 36: 407-477 (1972)), but the glutaminyl and diaminopimelyl residues in the peptide are amidated (Lederer, E., Pure Appl. Chem. 25: 135-165 (1971)). The peptidoglycan of M. leprae differs from that of other mycobacteria in that the amino acid in position 1 of the peptide is glycine instead of L-alanine (Draper, P., O. Kandler, and A. Darbre., J. Gen. Microbiol. 133: 1187-1194 (1987)). As a whole, the mycobacterial peptidoglycan exhibits a high degree of interpeptide crosslinking, primarily through DAP:DAP crosslinks in addition to the DAP:Ala crosslinks more commonly seen in other bacteria (Wietzerbin, et al., Biochemistry. 13: 3471-3476 (1974)). In relation to other components of the mycobacterial cell envelope it is known that the mycolyl-arabinogalactan is covalently attached to the peptidoglycan via a unique disaccharide phosphodiester linkage, forming the mycolyl-arabinogalactan-peptidoglycan complex (mAGP) (Besra, et al., Biochemistry. 34: 4257-4266 (1995), McNeil, et al., J. Biol. Chem. 265: 18200-18206 (1990)).
DAP biosynthesis is central in the structure of the mycobacterial peptidoglycan. DAP is neither produced or required by humans, and thus the DAP biosynthetic pathway is an attractive target for the development of anti-bacterial drugs. DAP auxotrophs of virulent M. tuberculosis might prove to be attenuated and therefore, potential live-vaccine strains.
DAP is synthesized by bacteria via the aspartate amino acid family pathway (Umbarger, H. E., Ann. Rev. Biochem. 47: 533-606 (1978)). This family is comprised of methionine, threonine, isoleucine, and lysine, amino acids whose carbon skeletons are primarily derived from aspartate. L,L-DAP, or its isomer meso-DAP are intermediates from this pathway used for peptidoglycan synthesis in some bacteria, while meso-DAP is the direct precursor to lysine in all bacteria (Umbarger, H. E., Ann. Rev. Biochem. 47: 533-606 (1978)). An aspartokinase enzyme, encoded by the ask gene in mycobacteria, catalyzes the first step in the aspartate family pathway (Cirillo, et al., Molec. Microbiol. 11: 629-639 (1994)). Earlier attempts at allelic exchange of the wild type chromosomal ask gene with a disrupted ask allele in M. smegmatis to obtain mutants auxotrophic for Met, Thr, and DAP were unsuccessful, suggesting that disruption of ask is lethal to this organism even when the products of the aspartate pathway are present in the culture medium (Cirillo, et., J. Bacteriol. 173: 7772-7780 (1991)). Since DNA recombination in mycobacteria is poorly understood, and the failure to obtain a gene disruption is not an absolute measure of the essentiality of that gene, a test to determine if ask is needed.
Accordingly, there exists a need to determine the essentiality of particular genes of mycobacteria in the biosynthesis of the peptidoglycan of the mycobacterial cell wall, as well as in the biosynthesis of other proteins. Once the essentiality of particular genes in these pathways is determined, the need becomes evident for the development of auxotrophic strains of mycobacteria containing mutations in these genes. Mutant mycobacterial strains are required for the biochemical analysis of mycobacteria. As M. tuberculosis is an airborne pathogenic mycobacterium, a mutant strain of M. tuberculosis that is safe to use in experiments in normal laboratory conditions is extremely desirable. Most significantly, mutant mycobacterial strains have potential therapeutic uses in the prevention and treatment of mycobacterial related diseases and conditions, and more specifically, can be used to deliver vaccines. In addition, there is a need for mutant mycobacterial strains to be developed and used in drug screening processes.